Method for forming volume independent test device

ABSTRACT

Volume independent test device and method for forming same, covering the top of reagent matrices with a water impermeable or semipermeable coating or membrane having spaced openings of limited size which permit liquid to pass through the coating or membrane and contact the underlying reagent matrix until the matrix becomes saturated are disclosed.

This is a division of application Ser. No. 746,767, filed June 20, 1985,now U.S. Pat. No. 4,647,430, patented Mar. 3, 1987.

FIELD OF THE INVENTION

The present invention relates to volume independent test devices and,more particularly, the present invention relates to method of forming avolume independent solid state test strip by covering the top of thereagent matrices with a water impermeable or semipermeable coating ormembrane having spaced openings of limited size which permit liquid topass through the coating or membrane and contact the underlying reagentmatrix until the matrix becomes saturated.

BACKGROUND OF THE INVENTION

The art of analytical chemistry has been greatly advanced sincebiochemistry began emerging as a primary scientific frontier, requiringincreasingly sophisticated analytical methods and tools to solveproblems. Likewise the medical profession has lent impetus to the growthof analytical chemistry, with its desiderata of both high precision andspeed in obtaining results.

To satisfy the needs of the medical profession as well as otherexpanding technologies, such as the brewing industry, chemicalmanufacturing, etc., a myriad of analytical procedures, compositions andapparatus have evolved, including the so-called "dip-and-read" typereagent test device. Reagent test devices enjoy wide use in manyanalytical applications, especially in the chemical analysis ofbiological fluids, because of their relatively low cost, ease of use,and speed in obtaining results. In medicine, for example, numerousphysiological functions can be monitored merely by dipping a reagentstrip test device into a sample of body fluid, such as urine or blood,and observing a detectable response, such as a change in color or achange in the amount of light reflected from or absorbed by the testdevice.

Many of the "dip-and-read" test devices for detecting body fluidcomponents are capable of making quantitative or at leastsemiquantitative measurements. Thus, by measuring the response after apredetermined time, an analyst can obtain not only a positive indicationof the presence of a particular constituent in a test sample, but alsoan estimate of how much of the constituent is present. Such test devicesprovide the physician with a facile diagnostic tool as well as theability to gauge the extent of disease or of bodily malfunction.

Illustrative of such test devices currently in use are productsavailable from the Ames Division of Miles Laboratories, Inc. under thetrademarks CLINISTIX, MULTISTIX, KETOSTIX, N-MULTISTIX, DIASTIX,DEXTROSTIX, and others. Test devices such as these usually comprise oneor more carrier matrices, such as absorbent paper, having incorporatedtherein a particular reagent or reactant system which manifests adetectable response, e.g., a color change, in the presence of a specifictest sample component or constituent. Depending on the reactant systemincorporated with a particular matrix, these test devices can detect thepresence of glucose, ketone bodies, bilirubin, urobilinogen, occultblood, nitrite, and other substances. A specific change in the intensityof color observed within a specific time range after contacting the testdevice with a sample is indicative of the presence of a particularconstituent and/or its concentration in the sample. Some of these testdevices and their reagent systems are set forth in U.S. Pat. Nos.3,123,443; 3,212,855 and 3,814,668.

Thus, it is customary for reagent test devices to contain more than onereagent bearing carrier matrix, in which each reagent bearing carriermatrix is capable of detecting a particular constituent in a liquidsample. For example, a reagent test device could contain a reagentbearing carrier matrix responsive to glucose in urine and another matrixresponsive to ketones, such as acetoacetate, which is spaced from, butadjacent to, the glucose responsive matrix. Such a product is marketedby the Ames division of Miles Laboratories, Inc. under the trademarkKETO-DIASTIX. Another reagent test device marketed by the Ames Divisionof Miles Laboratories, Inc., N-MULTISTIX, contains eight adjacentreagent incorporated matrices providing analytical measurement of pH,protein, glucose, ketones, bilirubin, occult blood, nitrite, andurobilinogen.

For some assays it has been found that test devices cannot be used inthe normal way by simply dipping the test device into a sample orsolution to be measured. For such assays the amount or volume of thesample which contacts the reagent test device is very critical and forsuch assays a very precise amount of sample must be applied to thereagent matrix each time an assay is performed in order to achieveconsistent results. For such assays the development of a volumeindependent test device in which the amount of sample which comes incontact with the reagent matrix remains constant each time an assay isused would be extremely important since it would overcome problems ofinconsistent results due to differences in the amount of samplecontacted with the reagents in a matrix. The present invention isprimarily directed to achieving constant sample loading per unit area ofa reagent matrix and thus a truly volume independent test device.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a volume independenttest device.

Another object of the present invention is to provide test devices inwhich a constant sample loading per unit area can be achieved.

Still another object of the present invention is to provide a method ofproducing a volume independent solid phase test device.

In accordance with the present invention, the reagent matrix of aconventional dip-and-read test device is covered on at least its topsurface with a water impermeable or semipermeable coating or membranehaving in excess of three openings which permit liquid to pass throughsaid membrane to the underlying reagent matrix until said matrix becomessaturated. The percentage of the surface area taken up by the openingscan range from a lower limit of about 0.01 percent to an upper limit ofabout 5 percent.

In use the amount of sample applied to the exposed surface of thecoating or membrane becomes unimportant. Sample placed on said coatingor membrane passes through the openings until the underlying reagentmatrix pad is saturated. Upon pad saturation, the remainder of thesample remains on top of the coating or membrane and is prevented fromreacting with reagents in the matrix pad, the coating or membrane thuseffectively becoming a barrier to further contact between remainingapplied sample and components of the reagent matrix. Accordingly, theresulting test device achieves constant loading of sample per unit arearegardless of the amount of sample applied to the test device and avolume independent test device is obtained.

The volume independent test devices of the present invention and theprocess for forming such test devices are suited to a broad range ofvolume sensitive solid phase test devices which are based primarily onabsorbent matrices such as paper. These include assays for glucose, AST(cholesterol), ALT (phenobarital), theophylline and other immunochemicalassays.

BRIEF DESCRIPTION OF THE DRAWINGS

Other and further objects, advantages and features of the invention willbe apparent to those skilled in the art from the following detaileddescription thereof, taken into conjunction with the following drawingsin which:

FIG. 1 is a diagrammatic side view, in cross-section, of a test deviceillustrating an embodiment in accordance with the present invention;

FIG. 2 is a diagrammatic side view, in cross-section, illustratinganother embodiment of a test device in accordance with the presentinvention;

FIG. 3 is graph depicting the performance of a normal hexokinase glucosetest device of the prior art; and

FIG. 4 is a graph illustrating the performance of a volume independenthexokinase test device in accordance with the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to the drawings, test devices in accordance with the presentinvention are illustrated in FIGS. 1 and 2. These figures illustratereagent test devices which comprise reagent matrix areas attached to asubstrate. The reagent pad or matrix material 10 (in FIG. 1) and 20 (inFIG. 2) can be formed from any suitable material. U.S. Pat. No.3,846,247, for example, teaches the use of felt, porous ceramic materialand woven or matted glass fibers. Additionally, U.S. Pat. No. 3,552,928teaches the use of wood, cloth, sponge material and argillaceoussubstances. The use of synthetic resin fleeces and glass fiber felts ascarrier matrix is suggested in British Patent No. 1,369,139. AnotherBritish Patent No. 1,349,623, proposes the use of light permeablemeshwork of thin filaments as a cover for an underlying paper matrix.Polyimide fibers are taught in French Patent No. 2,170,397.Notwithstanding these suggestions, however, the material predominantlyused in the art as a carrier matrix and that which is especially usefulin the present invention is bibulous paper, such as filter paper.

The reagent system which is present depends on the particular analyte tobe measured and is within the skill of those in the art. Normally thereagents are impregnated into the carrier matrix prior to the attachmentof the carrier matrix to a suitable substrate material.

The substrate (12 in FIG. 1 and 22 in FIG. 2) can be formed from anysuitable material such as cellulose acetate, polyethylene,terephthalate, polycarbonate and polystyrene. In the embodimentsillustrated in FIGS. 1 and 2 substrates 12 and 22 extend for somedistance beyond the overlying reagent matrix area 10 and 20,respectively. This permits an end of the test device opposite thatcontaining the reagent matrix to be used as a handle for convenience inperforming an assay. In a preferred embodiment the reagent matrix (10and 20) is attached to a substrate 12 and 22, respectively, which iscomposed of polystyrene (Trycite, available from Dow Chemical Company)using double faced adhesive tape known as Double Stick, available fromthe 3M Company.

Covering the top and opposite ends of reagent matrix 10 in FIG. 1 is acoating or membrane 14 which is either impermeable to aqueous fluids orsemipermeable. This coating or membrane 14 actually adheres to not onlythe reagent matrix 10 but also substrate 12 and can be used to maintainreagent matrix 10 in place attached to substrate 12. Coating or membrane14 normally has a thickness ranging from about 0.005 mm (millimeters) toabout 2 mm and preferably from 0.1 mm to 1 mm. The membrane ispreferably made of a transparent or translucent material. However, ifreadings are taken from the bottom of the test device through thesupport material 12 then the overcoat layer or membrane 14 can beopaque. Suitable transparent or semitransparent materials for membrane14 include gelatin coatings, Scotch Tape, polyethylene, waxes plastics,silicone, rubber, etc.

Waxes which are especially useful for use as an overcoat barrier layerare those which are thermoplastic, water repellent, smooth in texture,nontoxic and have freedom from any objectionable odor or color. Majortypes of waxes which can be employed include natural waxes, such asanimal wax, beeswax, spermaceti, lanolin, shellac wax; vegetable waxes;such as carnauba, candelilla, bayberry, sugar cane; mineral waxes, suchas fossil or earth waxes, including ozocerite, ceresin, montan; andpetroleum waxes, such as paraffin, microcrystalline, petrolatum; as wellas synthetic waxes such as ethylenic polymers and polyolether-estersincluding Carbowax, sorbitol and chlorinated napthalenes such as Halowaxand other hydrocarbon waxes. A preferred wax is the WW0404 wax from H.B. Fuller Company of Kalamazoo, Mich., which has the followingcharacteristics: Melting point (ASTM D127) 82° C.±4%, hydrophobic,inert, bendable and not tacky when dry. The congeal point (ASTM D938) is76° C.±4%, viscosity (Brookfield Thermocal) is 17.5 cps 93° C., andcolor (ASTM D1500) is 1.0 Saybolt.

Gelatins which are especially useful for use as an overcoat barrierlayer are those with small pore size to hinder diffusion to the maximalextent possible. Skin, bone, acid and alkaline processed gelatin from avariety of sources can be used. These include, but are not limited to,Agfa H9, Agfa H7/8, Agfa 2KN-407 (available from Bayer AG) and Fishergelatin. Small pore size is generally obtained by using gelatinconcentrations in the the 5-20 percent range and by using 1 percent ormore of crosslinkers such as Xama-7 (Cordova Chemical Company, NorthMuskegon, Mich.), Agfa SOB-2402, methoxyethyl-bisvinylsulfone and otherprotein crosslinking agents.

Any conventional coating methods can be employed to apply coating ormembrane 19 including direct and indirect gravue coating, transfercoating, and other coating methodologies which will produce coatings ofthe desired thickness and other characteristics mentioned. If thecoating or membrane 14 does not readily adhere to matrix 10 (orsubstrate 12, in the case of FIG. 1) various adhesives can be applied tojoin membrane 14, matrix 10 and/or substrate 12 together.

For conventionally dimensioned reagent test devices the dimensions ofsurface 18 of coating or membrane 14 will normally be 0.5 by 1 cm.Surface 18 will contain from 3 to about 300 holes, approximately thesize of pin holes in either a regular or irregular spaced configurationsuch that the holes or openings 16 are fairly uniformly distributedacross the total area of the upper surface 18 of membrane 14.Preferably, the number of holes is in the order of magnitude of 30 andthese holes, which can have a diameter from 0.1 mM to 1 mM, and, intotal, occupy from about 0.01 percent to about 5 percent and generallyaround 1 percent of the total surface area of surface 18. These openingspermit sample liquid 19 to flow through membrane or coating 14 intoreagent matrix 10 until the reagent matrix becomes saturated.

The number of holes extending through the overcoat can thus be variedwithin certain limitations. If the density of the holes is too few thensample will penetrate into the reagent matrix at too low a rate and ifthe density of the openings is too high then the penetration into thereagent matrix area could prevent the overcoat from acting as a barrieronce the reagent matrix becomes saturated and therefore defeat thepurpose of the separation.

It has been demonstrated that it is essential to have the holesextending through the coating or barrier material also extend into thereagent matrix material for some distance. Normally, penetration in therange of from 10 percent to 100 percent and preferably from 20 percentto 50 percent into the reagent matrix area is sufficient. Unless theholes penetrate not only the overcoat but also penetrate slightly intothe reagent matrix essentially no sample take-up is possible. This meansthat normally the overcoat will be applied to the reagent matrix andthen the holes are formed so as to penetrate the overcoat into thereagent matrix material.

Typically about 30 microliters of sample 19 is applied to a test devicein accordance with the present invention and the reagent pad or matrixabsorbs 20 to 25 microliters of sample. Openings 16 combined with theabsorbent material comprising reagent matrix 10 create a wicking actionwhich serves as the driving force for the transport of sample 19 intoreagent matrix 10 through opening 16. This wicking action acts toquickly pull sample through the holes until the matrix 10 is saturated.Once the matrix is saturated the wicking action stops since there is nodriving force to cause sample to flow through the holes and excesssample remains on top of membrane 14 isolated from the reagents inmatrix 10 by the barrier imposed by membrane 14.

Thus, in accordance with the present invention the amount of sampleplaced onto the reagent test device is in excess of that required tosaturate the reagent pad. When the reagent pad becomes saturated furtherflow of sample stops and the remainder of the sample remains separatedfrom the reagent pad by the membrane or coating layer and does notthereby affect the reaction.

As seen in FIG. 2 it is not necessary for the membrane or coating on topof the reagent matrix area 20 to actually cover one or more sides of thematrix material. In FIG. 2 reagent matrix 20 is attached to substrate 22and the overcoat layer 24, ranging from 0.005 millimeters to about 2millimeters in thickness and preferably from between about 0.01 mm andabout 1 mm thickness, covers just the top surface of reagent matrix area20. The openings 26 in layer 24, similar to openings 16 in FIG. 1, takeup an area ranging from about 0.01 to about 5 percent of the totalsurface 28 of membrane 24. The configuration of the openings 26 in FIG.2 differ slightly from the V-shaped openings 16 illustrated in FIG. 1.Notwithstanding, as in FIG. 1 the same volume independency is achievedwhen sample 29 is applied to the top surface 28 of the resulting reagenttest device of FIG. 2. When reagent matrix 20 becomes saturated layer 24acts as a barrier which prevents any further sample 29 from contactingreagent matrix 20.

The volume independent test devices of the present invention can beformed by any suitable method with the openings 16 and 26 formed in thecovering film or membrane layer either before or after (but normallyafter) membranes 14 and 24 have been applied to cover the top of reagentmatrix areas 10 and 20, respectively. For example, one procedure forforming the test devices of the present invention is to apply a coatingover the reagent matrix areas and then subject the coating to a rollerhaving a series of needles arranged in a set pattern which causepenetration of the coating and the underlying matrix material to achieveopenings ranging from about 0.01 to about 5 percent of the total surfacearea covering the reagent matrices.

As previously indicated, the openings occupy only a small portion of thetotal surface area with the number of openings ranging from about 3 toabout 300. Typically 10 to 50 openings are present and are of such asize that the openings prevent liquid from flowing through without adriving force in addition to gravity. The size of the openings thus issuch that the surface tension prevents liquid from flowing through theholes unless a driving force caused by the wicking action of theunderlying reagent matrix brings about the transfer of fluid from thesurface of the overcoat into the reagent matrix area.

Accordingly, the present invention distinguishes over prior art,including U.S. Pat. No. 3,802,842 which describes a test device havingmeshwork employed to hold the reagent matrix in place and protect itsince, as indicated in that patent, the open surface of the meshworkranges from 30 to 80 percent of the total surface area and the meshworkdoes not and can not serve as a barrier by which a volume independenttest device is formed. The number, size, extent and purpose of theopenings are diametrically opposed to the concept of a sample barrier.

The present invention also differs from devices such as those disclosedin U.S. Pat. No. 3,690,836 which are used primarily for studyingbiological reactions. These devices comprise a sandwich of sheets sealedtogether with one opening for the introduction of the biologicalmaterial into the sealed chamber and another opening to permit theescape of air.

The present invention also differs from multilayered test devices whichincorporate filtering layers and/or spreading layers into the structure,such as set forth in U.S. Pat. No. 4,256,693. The normal spreading ofsuch devices is completely porous and is designed to facilitate theintroduction of and spreading of sample throughout the reagent matrixuniformly. Accordingly, the spreading layer does not and could not actas a barrier layer.

The following Examples are given for the purpose of illustrating thepresent invention.

EXAMPLE I

Whatman 31-ET paper was impregnated with chemical components for anenzymatic hexokinase based test for serum glucose, as set forth in C.Wilsey, E. Kurchacova, R. Ide, "A Solid-Phase Reagent Strip Test for theColorimetric Determination of Serum Glucose on the SERALYZER®Reflectance Photometer" Clinical Chemistry, Vol. 30, No. 6, p. 969(1984) and the paper was then dried. After drying, the paper was coatedwith a 10 percent gelatin--1 percent XAMA (cross-linker from CordovaChemical Company, North Muskegon, Mich.) layer using a Number 34 MeyerRod and indirect transfer coating. After the coating had hardened thesurface was perforated with 0.25 mm (10 mil) diameter wires to create aregular array of pin holes with 1.6 mm (1/16th inch) spacing between theholes. The resulting reagent matrix material, covered with its overcoat,was then applied to Trycite using double stick adhesive and theresulting reagent cards were slit into test strips by conventionalmeans.

FIGS. 3 and 4 compare the strip performances between a normal hexokinaseglucose test strip (FIG. 3) of the prior art and a volume independenthexokinase glucose test strip made in accordance with this Example (FIG.4). Each test device was given a low [78 milligrams per deciliter(mg/dl)] and a high (277 mg/dl) concentration of glucose in 25microliters, 30 microliters, 35 microliters and 40 microliters samplevolumes. The reflectivities of the resulting test devices were thenobserved every 5 seconds using a Seralyzer® photometer available fromthe Ames Division of Miles Laboratories, Inc., making the readings at620 nanometers. The test strip reflectivities (R) were converted to alinear function of sample concentration L(R) by applying the followingformula: ##EQU1## As seen from FIG. 3, the reaction profiles of normalhexokinase glucose test devices vary greatly. By contrast, there isalmost no variation in the reactivities of the volume independentglucose test devices over the 60 seconds of the assay.

Thus, the performance of the test device of this example is effectivelyvolume independent between the 25 to 40 microliter sample range over thecourse of one minute. The presence of excess material on top of thereagent matrix can be minimized optically by using an integrating sphereor, alternatively, excess sample can be wiped off and the test deviceobserved visually or instrumentally, with or without an integratingsphere. Accordingly, the present invention removes one of the majorproblems involved in using test devices which are volume dependent. Evenwith automated pipetting it is extremely difficult to obtain completeand consistent pipetting of material and these problems are compoundedwhen manual pipetting is attempted. Test devices in accordance with thepresent invention are especially desired for end point assays since ratetests are nearly volume independent.

From the foregoing, it will be seen that this invention is well adaptedto attain all of the ends and objects hereinabove set forth, togetherwith other advantages which are obvious and inherent to the system. Thevolume independent test device of the present invention is easy to useand easy to prepare. In use, an operator does not have to be precise inthe amount of sample applied to the test device. Moreover, it is notnecessary to use calibrated pipettes to apply sample to the testdevices. Any system capable of applying sample to the test device,including eye droppers, can be employed. The only requirement is thatmore sample be applied than is required to saturate the pad or thereagent matrix. Normally, the pad or reagent matrix becomes saturatedwithin seconds. Thus, the present invention has the advantages ofconvenience, simplicity, relatively inexpensiveness, positiveness,effectiveness, durability, accuracy and directness of action. Theinvention substantially overcomes problems associated with end pointassays and other volume dependent tests by removing a problem which hasexisted for a long period time in connection with solid state testdevices. The invention provides a very effective, simple and inexpensiveway of eliminating the volume dependency of such test devices. Moreover,the present invention provides a method of performing volume independenttest devices which is compatible with existing techniques and methodsfor forming reagent test devices. It will be apparent that even thoughonly one reagent matrix is shown in FIGS. 1 and 2, test devices could beformed in accordance with the present invention which have multiplereagent matrices present on the same substrate.

Obviously, many other modifications and variations of the invention ashereinbefore set forth can be made without the departing from the spiritand scope thereof, and therefore only such limitations should be imposedas are indicated by the appended claims.

What is claimed is:
 1. A method of forming a volume independent testdevice which comprises coating a reagent containing matrix with a layerof water impermeable or semipermeable material and then forming holes insaid layer such that said holes occupy from about 0.01 percent to about5 percent of the total surface area of the top surface of said material,wherein said holes extend a distance of 10 to 100 percent into saidmatrix.
 2. The method of claim 1 in which said holes are formed using aroller.
 3. The method of claim 1 in which said holes are formed using apunch.